Method and means for assaying biological factors demonstrating quantal response

ABSTRACT

Method and apparatus for sampling a system to obtain a series of aliquots from the system in a single step and for inoculating a correlated series of sample-accepting zones in a single procedural step, each zone including indicator means for manifestation of organism growth in the zone, whereby quantal response and a measure of the concentration of biologically active entities contained in the system being analyzed may be conveniently and accurately determined.

22 Filed:

ilit

States Patent [191 Kaye METHOD AND MEANS FOR ASSAYING BIOLOGICAL FACTORS DEMONSTRATIN QUANTAL RESPONSE [76] Inventor: Saul Kaye, 1001 Ridge Ct.,

Evanston, 111. 60202 Apr. '10, 1972 211 Appl. No.: 242,707

[52] US. Cl. 195/1035 R, 195/140 [111 3,787,290 51 Jan. 22, 1974 2,904,474 9/1959 Forg 195/139 3,509,026 4/1970 Sanders.... 195/1035 R 2,771,398 11/1956 Snyder 195/1035 R 3,001,914 9/1961 Andersen 195/1035 R" 2,956,931

10/1960 Goldberg l95/l03.5 R

Primary ExaminerA. Louis Monacell Assistant Examiner-R. B. Penland Attorney, Agent, or Firm-Kegan, Kegan and Berkman [5 7] ABSTRACT Method and apparatus for sampling a system to obtain a series of aliquots from the system in a single step and for inoculating a correlated series of sample-accepting zones in a single procedural step, each zone including indicator means for manifestation of organism growth in the zone, whereby quantal response and a measure of the concentration of biologically active entities contained in the system being analyzed may be conveniently and accurately determined.

11 Claims, 12 Drawing Figures PAIEMIEDJANEZW 3.787, 290

| 512 256 I28 64 32 I6 8/ 4) 2 1 1 WWWWWWTWWWWW W W METHOD AND MEANS FOR ASSAYING BIOLOGICAL FACTORS DEMONSTRATING QUANTAL RESPONSE BACKGROUND OF THE INVENTION Field of the Invention This invention is directed generally to, though not limited to, the measurement of water and food quality, environmental sanitation, pollution control, and public health aspects of microbiology. More particularly, the method and apparatus of the invention find utility in determining the number of viable microorganisms of various types which are present in various physical media or systems including liquids and solids, on surfaces, and in the air. Typical applications in which such determinations are important, and in which this invention may be usefully employed are:

liquids: drinking .water supplies; streams, wells, water sources; effluent and waste waters; public and private bathing facilities; recirculating water supplies such as cooling towers and pasteurizers; paper mill process waters; oil-field flooding waters; milk, beer, other beverages; ice cream; liquid or creamed food products; cosmetic or pharmaceutical liquids, creams, jellies, pastes, ointments; blood; urine; spinal fluid, etc.;

solids: foods; drugs; cosmetics; paper; fabrics and dressings, etc.;

surfaces: patients skin, wounds, dressings; hospital floors, ceilings, walls, sinks, furniture; linens and uniforms; surgical wards, gloves; hospital equipment such as respirators, incubators, tents; sterile assembly and filling areas inpharmaceutical and aero-space facilities; food processing plants and equipment; food chillers and refrigerators; finished or in-process foods such as chicken, fish, meat in plant'or in market; frozen foods during processing, storage, and sale; dairy utensils, equipment, and containers; bulk pasteurizing machinery for milk and beer; utensils; glassware, dishes and surfaces in restaurants, bars, hospitals, schools, nursing homes, public eating establishments, etc.;

air: operating suites and special patient isolation rooms such as incubators, oxygen tents, burn wards; hospital rooms and corridors; sterile and clean rooms ofdrug manufacturers, aero space facilities; dairies; breweries, food plants; chicken houses, milking barns, eviscerating plants, etc.

In addition to the above, the method and means of the invention can be used in such widely diverse fields as toxicology, immunology, pharmacology, plant pathology, virus research in any discipline where quantitative results depend upon the observation of a quantal" response. Many examples are found in the field of environmental sanitation, but the same principal and analogous devices may be used in determining the number of infective units in a virus or bacteriophage suspension; determining the sensitivity of patients to varying strength of ragweed pollen suspensions or other allergies; quantitative broth testing of antibiotic sensitivity of various bacterial species; potency of insecticides, attractants, sterilants in entomology, etc.

Relevant Theory and Procedures quantal appearance, i.e., produce an all-or-nothing reaction. For example, one judges whether a liquid is free of microorganisms by introducing an aliquot or a portion of the liquid into suitable nutrient media. If after an appropriate incubation time, there is no evidence that growth has occurred, we define that portion of the liquid as sterile. It is possible that if one were to inoculate a larger portion or aliquot of the liquid into nutrient media, it would contain a microorganism, and thus cause the nutrient to show signs of bacterial multiplication. If a liquid system isknown to contain microorganisms, it is always possible to dilute the system with sterile liquid to such an extent that given aliquots of the dilution contain no viable microorganisms, and inoculating such aliquots into culture media will produce no quantal response.

In analogous fashion, the potency of toxic or thera peutic materials can be assayed by diluting them to the extent that they cause no response (either illness or cure) in the organism used as assay. In immunological testing, the material or fluid being assayed is diluted serially, and observation is made of the ability of each successive dilution to produce the particular serological or immunological reaction which is of interest.

The method of enumerating the number of active entities, or the potency of a preparation, depends upon diluting the preparation to the point where successive dilutions respectively do and do not produce the quantal response in the test system. There have been hundreds of published investigations, both mathematical and experimental, into the statistical validity of this method of enumerating active entities, and standard methods have been developed for determining not only the bacterial population or the biological potency of preparations which produce a quantal response, but for obtaining as well estimates of the probable errors involved in using the several statistical parameters thus obtained.

In order to clarify the basic concept of this invention, a brief summary will be given of some biological test methods which depend upon the observation of a quantal response. I

'Toxic, infectious, or curative potency is determined by a quantal method which involves diluting the agent of interest and applying the dilutions to appropriate test systems" or animals. The number of animals dying, infected, or cured, and the total number treated with each dilution are observed. Statistical formulas are then applied to the dilutions and the observed responses, and an index called L.D. l.D. or ED. (Lethal Dose, Infectious Dose, or Effective Dose in 50 percent of the test systems or animals) is calculated. This is the dilution of the product which will cause the observed quantal response to occur in only half of the assayed animals, while the remaining half are unaffected. .l.H. Gaddum, British Medical Research Council Report No. 183, London, 1933; L. .1. Reed and .l. Muench, Amer. J. Public Health, 27, 493-497 (1938).

In the case of concentration of viable microorganisms, the statistical term, Most Probable Number (MPN has been introduced to describe the parameter which can be derived by diluting the sample serially and observing which dilutions do not produce growth in nutrient media, and which do. (H. O. Halvorson and N. R. Ziegler, J. Bacteriology, 25, l0l-l2l, 1933; W. G. Cochrane, Biometrics, 6, -116, 1950.) In this method, instead of diluting the fluid whose bacterial concentration is desired, one plants replicate samples of different volumes of the fluid in nutrient media and observes which samples produce evidence of growth, and which do not.

lmmunological tests are also based upon a quantal response. The serum or other biologically active material is diluted serially and each dilution assayed against a factor with which it produces some visible or determinable reaction. Such reactions, usually conducted in buffered physiological solutions or in gelled media, involve precipitations, agglutinations, coagulations, or similar visible phenomena.

Three features distinguish the quantal-response method of enumeration. 1. Dilution the preparation containing the active ingredient is diluted in a continuous series and each dilution is assayed by whatever means are appropriate to observe the response. Dilutions may be two-fold, i.e. 1:2, 1:4, 1:8, 1:16, etc., or ten-fold, i.e. 1:10, 1:100, 1:1000 etc., or any other convenient ratio. The equivalent of a series of dilutions is using samples of different sizes taken directly from the material to be tested, i.e., ml, 1.0 ml, 0.1 ml. 2. Extinction each dilution must be tested until one is found where not all replicates give positive reactions. The most certain values, statistically, are those ob tained from dilutions which produce 50 percent positive and 50 percent negative results. 3. Replication the dilutions are prepared and tested in 3,5,8, or 10- fold replicates. Increasing the number of replicates improves the precision of the estimate of the population. (The mathematical background and basic tables are given by W. L. Stevens in Statistical Tables for Biological, Agricultural and Medical Research," R. A. Fisher and F. Yates, Edinburgh (1948), pp. 7, 8, Table Vlll- 2).

Direct Contact Methods for Enumerating Bacteria While the dilution method outlined above provides accuracy and precision, there are occasions where other methods of determining numbers of bacteria have been found to be useful. Since the present invention is applicable to such direct contact methods as well, a brief description of these methods follows.

Surfaces: A well-established method of determining the number of viable bacteria on horizontal surfaces is to pour molten nutrient agar-containing media upon the area to be examined and allowing the medium to incubate in situ (Hammer andOlson, Iowa State Coll. Agr. Station Bulletin 141, (1931)), or to transfer the hardened medium to a dish for incubation. (Angelotti, Foter, and Lewis: Food Research, 23 173-185 (1958)). Bacteria are transferred from the surface to the nutrient medium and develop into visible colonies in the latter. Probably the most used method of quantitating the number of bacteria on surfaces, however, involves ready-poured and hardened sterile nutrient materials which are pressed against the surface to be sampled and then placed in an incubator. Such devices have been reported in the following publications: Walter, Bacteriological Reviews, 19, 284-286 (1955); Seidal and Plaschke, Die Fleischwirlschaft, 10, 276-277 (1958); Forg, US. Pat. No. 2,904,474 (1959); Foster, Lancet 1,670-673, Mar. 26 (1960); Rubbo and Dixon, Lancet 2,394-397 Aug. 20 (1960); Hall and Hartnett, Public Health Reports, 79, 1,02l-1,024 (1964); Ten Cate, J. Applied Bacteriology, 28, 221-223 (1965); Forgacs, US Pat. No. 3,337,416 (1967). At least two devices based upon this principle have been introduced in the United States: the Rodac Plate by Baltimore Biological Laboratories, and the Monoflex Contact Plate by Hyland Laboratories. Some of the embodiments of the present invention can be used to improve the direct surface sampling method. I

Liquds: The direct contact method has also been used to enumerate the number of bacteria in liquid samples, and while it does not improve accuracy or precision, it does present a much simplified method of obtaining an approximate count of the viable organisms in the liquid. An example of such a device is the Oxoid Dip-slide described by Naylor and Guttman in J. Hygiene (Camb) 65, 367-371 (1967). Microscope slides are coated with nutrient agar and are dipped into samples of urine-and then incubated. Bacteria in numbers proportional to their content in the urine become attached to the nutrient surface and upon incubation develop into visible colonies. As will be seen, some embodiments of the present invention incorporate considerable improvement over this type of sampler.

Air: Direct methods for air sampling include the exposure of sterile nutrient surfaces for definite periods of time, followed by incubation of the plates. The present invention introduces a modification of the type of nutrient surface exposed and thereby produces advantages over existing methods.

Transfer Methods for Enumerating Bacterial Content of Surfaces To overcome some of the inconveniences of directly sampling surfaces, there have been described some methods which involve use of a sterile transfer device which is pressed against the surface and then pressed against a solid nutrient surface, transferring the viable organisms thereto. (Greene, Vesly and Keenan, J. Bact., 84, 188-189 (1962); Gundstrup, Health Laboratory Science, 5, 113-115 (1968)). The present invention has embodiments which are adaptable to this method of surface assay, as well.

The Most Probable Number (MPN) Method Brief mention has been made of the MPN method for determining the number of coliform organisms in drinking water supplies, but since the present invention is an extension of the principles involved therein, the method will be described more fully.

In one of its most-used forms, the MPN method requires; for each water sample, five capped, sterile tubes containing 10 ml. each of an appropriate sterile nutrient broth, made up to double-strength; five more sterile tubes containing 19 ml. each of single-strength nutrient broth, as well as a supply of sterile pipettes. To perform the test, the tubes are placed in a rack, and 10 ml. of the water being assayed are added to each of the five tubes containing 10 ml. of double-strength broth. One ml. portions of the water are added to the 19 ml. tubes, and 0.1 ml. portions are added to the 19.9 ml. tubes. All tubes are incubated, and examined after 24 or 48 hours for evidence of bacterial growth.

Standard tables have been developed for converting observed numbers of positive and negative tubes into an index called the Most Probable Number of bacteria per ml. of the water sampled. (American Public Health Association Standard Methods for the Examination of Water and Waste Water," 12th Ed., pp. 604-609, (1965). By way of concrete example, if the five tubes planted with 10 ml. each of water produced 5+re actions while the five containing 1.0 ml., and the five containing 0.1 ml. each gave no reaction, referring to the MPN table gives us a value of 23 organisms per'lOO ml of water. In addition the table tells us that 95 percent of the time, water giving these experimental results would have no less than 7 organisms per IOO ml, nor more than 70. I

Hundreds of such tests are conducted each day in public health laboratories, and so it will be appreciated that a considerable amount of sterile glassware and media are employed, and much time spent in preparing the materials and performing the manipulations required by this test. One object of this invention is to provide simple means for rapidly performing the operation of this test with less time, effort, materials and cost than are presently required.

GENERAL DESCRIPTION OF THE INVENTION This invention may be realized in many embodiments, some of which will be described in detail below. In general, a multiplicity of areas or volumes are provided which are in geometric ratio to one another, and these areas or volumes are furnished with nutrient materials or biochemically reactive substances or solutions which produce a quantal response in the presence of the entity being sought. Some embodiments contact or contain a series of different areas or volumes to be sampled and expose them to media where they will show a quantal response. Some embodiments pick up' the entity from a series of areas or volumes which are in geometric ratio to one another and transfer these (dilute them) to equal areas or volumes of nutrients or reagents, so that in effect a series of dilutions has been performed.

The purpose of the invention is to supply means for eliminating the necessity for performing dilutions or for planting replicate and different volumes or for sampling different areas individually. The means provided by this invention, to be described in detail below, in effect perform the dilutions or sample different volumes. Each compartment of the embodiment will produce a quantal or response and the number of+ and responses from any embodiment is directly translatable into the Most Probable Number of Bacteria, or the L.D. of a toxin, or an equivalent potency unit for .a serum or allergen.- This conversion is simpleand rapid and eliminates the need for mathematical computation by providing a special table for each embodiment. In drawing up such tables, consideration must be given to the specific size of the compartments, the geometric ratio used, the number of replicates of each area or volume, the specific media being employed, etc.

While the terms areas, volumes, compartments have been employed in this description, the ob-. ject of the invention can also be accomplished by trays, films, templates, patterns, absorbent materials, aggregations of bristles or pins, foams or projections or types of matter arranged as described. The teaching of this disclosure will make other modifications apparent to those skilled in the specific fields to which these embodiments are directed, but the invention is claimed to cover all specific embodiments which are made obvious in this disclosure.

DESCRIPTION OF THE DRAWINGS This invention will be described in greater detail with reference to the drawings and'to specific examples which are illustrative of rather than limiting the invention. In the drawings:

FIG. 1 is a top plan view of an .uncovered multicompartment system-sampling apparatus of the invention, including replicate cavities to accommodate a plurality of samples of different volumes;

FIG. 2 is a front elevational view of the sampler structure of FIG. I with surmounting cover in place;

FIG. 3 is a top plan view of a second embodiment of a multi-compartment system-sampling apparatus, the compartments being arranged as a radial array;

FIG. 4 is a elevational view of the sampler structure of FIG. 3;

FIG. 5 is a top plan view of a panel or slide coated to provide a series of discrete test zones or islands in replicates of various sizes;

FIG. 6 is a front elevational view of the of FIG. 5;

FIG. 7 is a front elevational view of a panel formed with depressions, cavities, or compartments adapted to retain a biologically-active-entity-growth response indicating medium;

FIG. 8 is a top plan view of a test film, panel or sheet provided with a series of discrete replicate areas responsive to indicate growth of any biologically active entity present;

FIG. 9 is a side elevational view of the test panel of FIG. 8 installed and supported in a protective sheath or envelope;

FIG. I0 is a front elevational view of a test panel or tray similar to that depicted in FIG. 8 but formed with depressions adapted to hold an indicating medium responsive to any biologically entity present;

FIG. 11 illustrates a test panel and a template to be applied thereto to delineate test zones; and

FIG. 12 is a front elevational view of an assembly for sampling in a single step, and transferring in a single step, a plurality of samples having a geometric ratio.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In accordance with the principles and practices of the present invention, at least two distinct embodiments are contemplated. These include the direct contact" and the transfer technique, each of which is described generally in the paragraphs below, and specifically by way of examples.

1. Direct Contact Embodiments In carrying out the direct contact techniques in accordance with the invention, the device or assembly used contains nutrient or biochemically active media, subdivided in areas or volumes which bear a geometric ratio to each other. The media contact directly the liquid, surface, or gas containing the entity being sought. This is accomplished by pouring the sample liquid into such compartments, or pressing a solidified reactive subdivided surface against the surface being sampled, and observing for quantal response in the test areas, zones, or volumes of the apparatus.

2. Transfer Embodiments An intermediary transfer device is used to contact the liquid, surface, or gas being assayed, picking up the entity which is soughLThe sampled material is then introduced into a nutrient or development or reaction medium wherein the quantal reaction is observed. Examples of suitable systems are those in which:

a. the transfer agent is uniform over its surface, but the nutrient or recipient device is subdivided in geometric ratio areas of volumes;

coated panel b. the transfer agent is divided into geometric ratio areas or volumes, but the recipient device containing reactants is divided into equal or equivalent areas or volumes (See FIG. l2);

c. A combination of the previous two types, for making successive geometric dilutions where the initial concentration of the entity being sought is great and high dilutions are necessary to achieve the necessary requirement that some of the series of growth or reaction compartments show no growth or reaction, while other do show such positive reactions.

DIRECT CONTACT EMBODIMENTS EXAMPLE I One structural embodiment of this invention, capable of performing the Most Probable Number assay for bacteria in a fluidis shown as a top plan view in FIG. 1, and in front elevation in FIG. 2. The assembly 20 includes a tray 22 of rigid but formable material which may be plastic sheet or film, paper or cardboard, glass or metal, formed with depressions cavities, compartments, or zones 24 of various volumes, which in the examples shown include five replicate volumes of 10, 1.0 and 0.1 ml. These cavities 24 serve to contain the water or other fluid being assayed, the numbers shown on FIGS. 1 and 2 represent the volume of each. The geometrical arrangement of the depressions on the film or sheet is purely arbitrary and not critical to the invention. The compartments may conveniently have the following dimensions and may contain the following weights of nutrients.

Volume Depth Diameter Weight of (ml) (cm) (cm) Broth Solids (mg) 2 2.53 200 l 1 L13 20 (l.l 0.2 0.80 2

It is emphasized that this is a arbitrary arrangement; any number of replicate compartments of any volume, achieved by combining any convenient depth and diameter or geometrical form of depression, can accomplish the same purpose, and any geometric ratio of volumes, e.g., 2, 4, 5, 10, may be used as desired. The only critical factors are that the volumes of the several sizes must be known, and they must be in geometric ratio to one another.

In each compartment 24 is placed that amount of the dried constituents 26 of a nutrient broth which will, when dissolved in the amount of water or other fluid contained in the depression, be reconstituted into a nutrient broth. Such broths usually contain about 2 percent solids, consisting often of A percent peptone, 1 percent yeast extract and /2 percent NaCl, but not limited to these ingredients or proportions. The nutrient broth may contain additives of many types, such as dyes or indicators which will change color when bacteria have multiplied to a given extent, thus amplifying or enhancing the usual observation of turbidity. Other chemicals may be added which will permit the growth or indicate the growth of only certain specific microorganisms, and in general the dry ingredients of any microbiological or biochemical test which is desired may be placed in the compartments. The quantity placed in each compartment or reaction zone will depend upon the volume of the compartment and the final concentration of anhydrous chemicals desired in the compartment. For the case ofa nutrient broth containing 2 percent solids, and the compartments of this specific example, the amounts of broth solids required are listed in the table above.

The dried ingredients may be weighed into each compartment, or they may be deposited in each compartment by filling each to capacity with the broth and then evaporating off the water (preferably under vacuum), leaving the exact weight of'broth solids in each compartment needed to form a reconstituted broth when water is admitted. Alternatively absorptive paper or other suitable material may be saturated with broth and then dried, and pieces containing the desired amount of broth solids added to each compartment.

The assembly 20 is completed by a cover 28 which may be manufactured of any convenient material, such as plastic, metal, etc. It may be simply a film which fits tightly over all the compartments or may have raised areas corresponding exactly to the compartments. The cover may or may not be attached to the tray 22 by a hinge 30. The cover 28 may carry an adhesive 36 laid down in a pattern so that when the tray is covered, the adhesive causes tight adhesion of the cover to the areas intervening between sample compartments. In other modifications, the cover may possess ridges or depressions which interlock with depressions or ridges in the tray, in order to seal each compartment off from all others on the tray.

In manufacturing the assembly, after the broth solids 26 are added to the tray compartments 22 asdescribed above, the cover 28 is put in place and the assembly sterilized. Steam sterilization may be used if the components will withstand sterilizing conditions; otherwise ethylene oxide gas or other sterilization means may be used. A tape or other closure may be required to maintain the sterility of the inside of the assembly, or the unit may be packaged in paper or plastic bags prior to sterilization. These processes of sterilization and protection of sterile goods are now well known, and any suitable means may be used.

To operate, the cover 28 is removed and the tray 22 dipped in the body of water to be tested, and the cover replaced. The purpose of the adhesive seal of cover to tray, or of the interlocking ridges and depressions, is to permit the assembly to be shaken after the sample is taken, to distribute the broth solids 26 through the water while keeping contents of each compartment separate from all others. The assembly 20 is then placed in an incubator at the temperature required for maximum development of the organisms which are of interest. Alternate means of loading the assembly with the fluid to be tested include pouring the liquid over the assembly, or holding it under the tap or outflow, then gently shaking the assembly to remove water from the surfaces other than the test zones or compartments 24. It may be desirable to treat those areas 32 in between compartments with a non-wetting material such as Teflon or Kel-F poly halogenated hydrocarbons or with an adhesive 34 which will repel water and serve as a seal to the cover.

There should be provided together with each assembly, printed on it or accompanying it, a Table of MPN drawn up specifically for the volumes, ratios and replicate numbers of the compartments of the apparatus. After the incubation is over, the number of compartments is counted, and the MPN index of the water is readable directly from the table.

EXAMPLE 1] The preceding assembly (FIGS. 1 and 2) employs three sample volumes, 'five replicates of each, with a maximum volume of 10ml. In many cases, where the A somewhat modified embodiment of this invention, though identical in function to that of Example II, is shown in FIGS. 3 and 4, drawn to about the same scale as FIGS. 1 and 2. In this embodiment, the compartments 40 of the tray 44 are arranged as concentric squares; they may also be'concentric cylinders or any other convenient shape. A single set of nine compartwater is of good quality, it is desirable to plant volumes 5 gzz giz j z 24 ml {0.0093 ml occluples an are? larger than ml (l-loskins, .l. K. & Butterfield C. T. J. 0 X 4 q h i Example h Amer. Water Works ASSOC. 1,1014109 (1935)). array described, conta ning five replicate sets, occupies Also the variance and standard deviation of the Most area 48 square mches' Example In ls more economical of incubator and storage space. Example pwbable Number lower the low er F geomemc l0 Ill is also filled with dehydrated broth 46, is equipped T betwee successwesfamplesj dllutlons h with a-cover 48, and is sterilized before use. It is easier m FSher & Yates P t so If It were prachcal h to handle than Example I or and provides advantages wouldhehlore prhclse to use h rather h 1 in manufacture and in rapidity of reading of turbid fold dhuhohs' Uslhg the twofold Sample rah), compartments. As previously described, the areas heever, one must extend the series of sample further to be tween the test Zones 40 may be treated i h a sure of covering the same concentration of bacteria in wettabie i l 5( the water sample. Thus, it would take eight consecutive two-fold dilutions to sample the same volume range EXAMPLE 1v covered by three successive ten-fold dilutions. Since '"j until now it has been burdensome to plant long series Another embodiment,.for determining the bacterial of samples differing by a factor of two, most of the procontent of liquids, is shown in plan section in FIG. 5, cedures for analyzing water use ten-fold dilutions and and in elevation in FIG. 6. In one of its form it consists thus lose some of the precision inherent in the method. of a glaSS 0 pl miCI'OSCOPC Slide 52 Coated with Furthermore, increased precision may be obtained by nutrient-containing agar 54 in selected zones or areas increasing the number of replicates of each sample 56. One application of such an embodiment is the deplanted. Practical considerations involving the number termination of the bacterial Content of urine, much as of sterile tubes required and the time involved in plantreported y Mackey and S Y i Med. Journal 2, ing many replicates have resulted in the adoption of the p' techmque descnbed procedures which are not as precise as possible. by Naylor and (J- Hyg.(Camb). 65, 36741 With the means afforded by this invention, however, (1967)) latter devlce commerclany avallable it takes no more time or material to plant successive through 3 two-fold dilutions than it would ten-fold, or to make ten T devlce 9 FIGS and 6 dlffers 9"? those replicates than it would to make three replicates. Read- Scnb ed,above m possessmg characteristic h of this invention, namely a series of separate replicate mg the results takes a little longer with more compartmerits, but the precision may be increased manyfold. areas or Zones 56 related to each other gee In a second embodiment of this invention, having niemcany' These shown as Shaded .areas on these Figures. Shown are SlX areas l X 1 cm, SIX areas Va X k recognizable advantages over the embodiment of Excm, and 15 areas 1/1 X cm Square, each ofwhich com ample I, there IS used an assembly cap l of h tains a layer of agar 54, of suitable thickness. The agar five replicates ofeach of nine sample sizes in geometric 40 may Standard nutrient agar or medium series. Again it is emphasized that this array s arbi- (Mackey and sandys, Brit Med. Journal 1, 1,1173 the Sizes a loc-ahohs of h bemg g'veh (1966)), or any other specialized or differential memerely for completehes,S h as hmhlhg dium desired. The agar medium 54 may also contain a sehsethat is necessary is that h Volumes be color indicator which will facilitate the observation of known, and that they exist in geometric ratio to one anany bacteriological l i li i other. The cfi ifi i t e m y of E p ll have Areas 60 in between those containing agar 54 may be the following properties: (for each volume there are t d i h. a ili or a fluorocarbon oth r five cavities) known anti-wetting coating 62 or the basic f lm mayre Vol. (ml) 24 l2 6 3 1.5 0.75 0.375 0.187 0.093 Ht. (cm) 2 2 1.5 1 l 0.5 0.25 0.25 0.25 on. (cm) 3.92 2.76 2 20 1.95 1.;s Hm 1 3s 1,3s 0 .gs 0.74

Other properties and features of the assembly are the sist water, so only agar-containing areas 56 pick up liqsame as those in FIGS. 1 and 2, i.e., nutrient broth inuid. gredients, in proportion to the capacity of the cavity, In operation, the device, prepared aseptically or ster are contained in each cavity; the assembly is filled by ilized after preparation, is dipped into a sample of liqdipping into water or pouring the water on it; and a uid, i.e., urine, whose bacterial content is to be detercover is provided which eals off each cgmpartment, mined. The slide 52 is returned to its original sterile Accordingly, no separate illustration is deemed neces- 60 comaiflel: is transported f l '4 a t y sary. The entire assembly is sterilized either by the where itrs incubated. After asultable incubation time, manufacturer or the user b f one counts the number of areas of each size in which growth has developed. Reference to a special MPN Ta- EXAMPLE lll ble, developed and calibrated just for this purpose, yields an index number which represents the bacterial concentration of the original liquid. Both Mackey and Sandys and Naylor and Guttman performed calibrations on their devices, correlating the number of colonies they obtained with the number of bacteria per ml of urine. The shapes of their devices differ, and they use different media, but for every 1,000 bacteria/ml of urine they obtain between 0.15 and 0.8 colonies per cm of exposed nutrient area. On the average, if their population contains 1,000/ml, they observe one colony developing in each 3.3 cm of area; 10,000/ml produce one colony in 0.33 cin 100,000/ml produce one colony in 0.033 cm The device portrayed in FIG. 5 provides areas of 1 cm, 0.25 cm and 0.0625 cm It is thus ideal for obtaining an index of population up to 50,000/ml; if higher concentrations are encountered, smaller nutrient areas may be provided. The use of smaller areas will enable populations greater than 150,000/ml to be counted without the occurrence of confluent growth which at present limits the devices available to the public.

It is emphasized that the need to count individual colonies no longer exists; with this device only the number of areas or zones containing any growth at all is counted. Reference to an appropriate Table of MPN gives the desired index of bacterial population.

In this example, the area of successive areas bear a four-fold relationship to one another, and there are six, six and replicates. No difficulties arise in constructing MPN tables where different numbers of replicates are used. (See for example, Standard Methods for Examination of Water and Waste Water, op. cit.)

The relationship of the MPN index (or the number of areas becoming to the number of viable bacteria in the liquid is determined experimentally for each specific sampling device, just as was done by Mackey and Sandys, and Naylor and Guttman (op.cit.).

EXAMPLE V An alternate configuration for Example IV is shown in elevation in FIG. 7. The nutrient agar areas 66 may EXAMPLE VI An alternate arrangement of areas of nutrient agar 80 and of non-wettable substrate 82 is shown in plan section in FIG. 8 and in front elevation in FIG. 9. Here we show eight replicates, of areas 25, 2.5, and 0.25 square cm, affording a ten-fold ratio, placed upon a nonwettable film or sheet 86. Just as the device of Example IV, this sterile array is dipped into the liquid to be sampled and returned to its sheath 88 and cover 90 for incubation. The film or sheet 86 is held away from the walls of the sheath by fitting through a slot 96 in the cover. The number of replicates, physical size, ratio, specific geometry, nutrient, etc., can be varied without departing from the spirit of the invention. On completion .of incubation, one records the number of areas and the number of areas, and refers to a MPN table to obtain an index of bacterial concentration in the liquid sampled. As indicated in .FIG. 9, the agar coated Zones 98 may be formed on both upper and lower surfaces of the test sheet 86.

The agar surfaces need not be laid upon a sheet or film, but may occupy depressions in a tray, as indicated in FIG. 10. The shaded areas represent the cross sections of agar-containing compartments or zones 104. A

pair of replicates of four geometrically related areas are shown in cross-section.

EXAMPLE VII In another embodiment of this invention, illustrated in FIG. 11, a plate, dish or tray [10 containing nutrient agar I12 is covered with a mask, tape, or template 116 which, in use, is tightly pressed against the agar surface. The template 116 may be rigid or flexible, and of any convenient thickness. It has the same or slightly larger dimensions as the tray 110, and possesses openings 120 whose replicated areas are in geometric proportion to one another. The template 116 may assume the forms illustrated in FIGS. 5, 8 or any equivalent geometry, where now the shaded areas which forrnrly represented nutrient agar now are understood to be voids in the template throughvwhich agar 112 from the tray is exposed.

This embodiment has the advantage of lower cost of manufacture, since a common unsubdivided tray may be used as the base for a numberof modifications, while any of a variety of inexpensive template can be furnished.

The sterile tray 110, with superimposed template 116 in place, is dipped into the liquid to be sampled. The template may or may not be removed after sampling. The tray 110 is covered and incubated for an appropriate time. Once again, only the number of each size of sampling surface is tabulated, and an estimate of the MPN is made by referring to a suitable table.

The common features of these examples need not be repeated. It is emphasized that by using these devices one is enabled, in effect, to perform a series of dilutions of the original liquid and to plate each dilution, without the use of pipettes, dilution tubes, or Petri dishes. The MPN figure obtained is as significant as the plate count obtained by the prior methods'described above. Further, the entire process can be performed in the field, or wherever the sample is taken, so the need to sterilize sample bottles and carrythern to the field location is obviated. Also eliminated is the often-noted phenomenon of an impurity or a nutrient in the water or the bottle inhibiting or stimulating the bacteria to produce a spurious count. Sometimes a refrigerator is used to slow down the inhibitory or multiplication process, or to keep water overnight or over weekends until the dilutions and plating can be performed. Using the devices of the invention described, one can start the incubation process immediately and thus obtain estimates of the bacterial population that correspond exactly to the state of the system at the moment the sample was taken. Another advantage of the devices described in this invention is that nearly all of the tedious and timeconsuming laboratory work is eliminated.

EXAMPLE VIII Direct Contact of Surfaces of giving as accurate a representation of bacterial population on the surface as can the Rodac or Monoflex Contact Plates, but with the advantage of sampling a larger area, and not requiring counting of individual colonies.

If the surface is not contaminated uniformly, the devices referred to can still be used, with the following modification. A sterile sponge is wetted with water, diluent, or nutrient broth and passed over the surface, wetting it thoroughly and rubbing and spreading surface contamination as uniformly as possible over the surface. The wetted surface is then sampled by the'device, and one now obtains results similar to those obtainable by the Swab-rinse method.

Besides its simplicity and accuracy, the method of surface sampling described has a unique advantage. Commonly, floors and other contaminated surfaces contain a few microorganisms known as spreaders, because their colonies grow rapidly and spread over the entire surface of a nutrient plate, obscuring and inhibiting all other growth. The presence of such spreaders renders other types of contact plates useless in many circumstances (Kundsin et al., J. Bacteriology 82, 619 (1961)). However, the devices of this invention are immune to the effect of spreaders because of the nonwetting demarcations separating growth areas, effectively isolating a spreader to the area in which it initiates.

Direct Contact with Air lf the devices of this invention are allowed to stand in the open air, uncovered, for any standard period of time, they can be used to obtain an index of the airborne microorganisms just as they can for liquids and surfaces. Alternatively, a stream of air can be forcibly directed against their nutrient agar surfaces so as to deposit the organisms contained in them upon the sur faces of various sizes.

. Transfer Embodiments The previous examples have all taught methods by which, in effect, different volumes or areas of liquids or surfaces to be examined could be sampled without actually performing dilutions. A second class of device covered by this invention includes means for performing geometric series of dilutions, by transferring the liquid being sampled, in different volumes, to other volumes of nutrient broth or biologically active liquid. The transfer device can itself be subdivided in geometrically related units, and the transfer made to equal volumes of fluid, or the transfer means can be subdivided equally and the transfer-made to liquids which have geometrically related volumes.

Geometric Transfer Unit Uniform Nutrient or Reactive Liquid Compartments FIG. 12 illustrates partly in elevation and partly in section a device which can be used to perform the enumeration process of the invention either on a solid or' over each group of bristles 142 represent either the relative volumes of liquid which will be picked up and retained by that group of bristles, projections, or capillaries, or the surface area occupied by the base of the bristles when it contacts a surface to be sampled.

in using this assembly to sample liquid for bacterial count, a multiplicity of each size bristle is provided. The sterile brush assembly 140 (sterilized by steam or by gas, whichever is more appropriate to the materials of construction) is dipped in the liquid, allowed to drain, and then placed over the tray assembly which contains sterile nutrient broth in the cups 126. After incubation, the number of compartments of each size is tabulated, and a table of MPN, based upon the series of 10 successive two-fold dilutions, and' the calibrated volumes picked up by each cluster, is referred to to yield a MPN- for the liquid.

When a surface issampled, a sterile liquid can be poured upon it, the device used as a brush for a standardized period of time, and the organisms picked up on each cluster than allowed to multiply in each compartment.

While this invention has been described with reference to preferred embodiments and procedures, it is evident that the invention is not limited thereto. Further modifications of the method and products disclosed herein which fall within the scope of the following claims will be immediately evident to those skilled in the art. To the extent that these changes and modifications are within the scope of the appended claims, they are to be considered a part of this invention.

What is claimed is:

l. A sampling apparatus operative to effect, in a single exposure to a system to be analyzed, a series of sample aliquots to provide quantal responses,

said apparatus including means defining a plurality of sample receiving zones for simultaneous exposure to the system to be analyzed, said zones including zones of different volumes of known geometric proportions,

biologically-active-entity-response indicator means functionally present in each of said sample receiving zones,

said indicator means being operative to produce in each said zones a quantal response to a biologically-active-entity being assayed.

2. The apparatus as set forth in claim 1 wherein said sample receiving zones comprise container means defining a plurality of individually separate and-distinct compartments each adapted to retain liquid samples of said system to be analyzed, and

closure means to seal each said compartments, to

preclude contamination thereof and to obviate intertransfer of liquid samples from one to another of said compartments,

thereby to ensure sample integrity of each said compartments.

3. The apparatus as set forth in claim 2 including a reaction medium disposed in each said compartments and responsive to a biologically-active entity present therein to provide a visual manifestation,

said medium being provided in each said compartments in an amount proportional to the volume capacity of respective each said compartments, whereby, upon dispersion of said medium in a volume of liquid defined by said compartments the concentration of medium in the resulting solutions is substantially the same from compartment to compartment.

4. The apparatus as set forth in claim 1 wherein said sample receiving zones constitute visually distinct, individually indetifiable areas disposed on panel means having said biologically-active entity indicator means contained therein,

said panel means being immersible in a fluid to effect exposure thereof to a system to be sampled for assay of biologically-active entities present therein to determine the concentration of such entities.

5. The apparatus as set forth in claim 1 wherein said means defining said sampling apparatus includes panel means,

said biologically-active-entity-response indicator means being functionally distributed on said panel means,

template means adapted contiguously to overlie said panel means,

said template means defining open windows, including windows of differing areas, delineating zonal areas through which said indicator means is ex posed concurrently with exposure of said panel means to a system to be sampled for assay of a biologically-active entity contained in said system.

6. The apparatus as set forth in claim 1 wherein said means defining a plurality of sample receiving zones comprises means constituting an array of physically separate,

discrete sampling means,

each said sampling means adapted to serve as a sample pick-up and transfer element,

each said sampling means of said array having a sample transporting and transferring capacity which is in a known geometric ratio with others of the sampling means in said array,

whereby exposure of said biologically-active-entityresponsive indicator means to said array is effective to establish a series of sample assay systems having known geometric relationships with each other with reference to a biologically-active entity to be assayed.

7. The apparatus as set forth in claim 1 wherein said sample receiving zones are present in a series of replicates to enhance statistical reliability of data obtained.

8. The apparatus as set forth in claim 1 wherein said biological]y-active-entity-responsive indicator means includes a nutrient medium for supporting the growth of a biologically-active entity to be assayed.

9. The apparatus as set forth in claim 1 wherein said biologically-active entity responsive indicator means includes a diagnostic reagent giving rise to a visible manifestation upon presence of a biologically-active entity being assayed.

10. The apparatus as set forth in claim 1 wherein size and geometry of said zones are such that some of said zones develop and others fail to develop manifestations of biological activity of entities present in the system to be analyzed.

11. The method of separating a unitary sample into a series of aliquots and quantitatively assaying any system including liquids, solids, surfaces and gases to determine the number of biologically-active entities present in said system, said method comprising the steps of delineating a specific system to be analyzed,

presenting to said system a sampling apparatus including means operative to extract and to retrieve from said system a series of aliquots in a single step,

subjecting said aliquots to controlled environmental parameters including time and temperature conducive to promote a manifestation of biologicallyactive entities present in said aliquots, and

examining said aliquots to determine the existence of indicia establishing the presence originally of biologically-active entities to ascertain the concentration of such biologically-active entities present 

2. The apparatus as set forth in claim 1 wherein said sample receiving zones comprise container means defIning a plurality of individually separate and distinct compartments each adapted to retain liquid samples of said system to be analyzed, and closure means to seal each said compartments, to preclude contamination thereof and to obviate intertransfer of liquid samples from one to another of said compartments, thereby to ensure sample integrity of each said compartments.
 3. The apparatus as set forth in claim 2 including a reaction medium disposed in each said compartments and responsive to a biologically-active entity present therein to provide a visual manifestation, said medium being provided in each said compartments in an amount proportional to the volume capacity of respective each said compartments, whereby, upon dispersion of said medium in a volume of liquid defined by said compartments the concentration of medium in the resulting solutions is substantially the same from compartment to compartment.
 4. The apparatus as set forth in claim 1 wherein said sample receiving zones constitute visually distinct, individually indetifiable areas disposed on panel means having said biologically-active entity indicator means contained therein, said panel means being immersible in a fluid to effect exposure thereof to a system to be sampled for assay of biologically-active entities present therein to determine the concentration of such entities.
 5. The apparatus as set forth in claim 1 wherein said means defining said sampling apparatus includes panel means, said biologically-active-entity-response indicator means being functionally distributed on said panel means, template means adapted contiguously to overlie said panel means, said template means defining open windows, including windows of differing areas, delineating zonal areas through which said indicator means is exposed concurrently with exposure of said panel means to a system to be sampled for assay of a biologically-active entity contained in said system.
 6. The apparatus as set forth in claim 1 wherein said means defining a plurality of sample receiving zones comprises means constituting an array of physically separate, discrete sampling means, each said sampling means adapted to serve as a sample pick-up and transfer element, each said sampling means of said array having a sample transporting and transferring capacity which is in a known geometric ratio with others of the sampling means in said array, whereby exposure of said biologically-active-entity-responsive indicator means to said array is effective to establish a series of sample assay systems having known geometric relationships with each other with reference to a biologically-active entity to be assayed.
 7. The apparatus as set forth in claim 1 wherein said sample receiving zones are present in a series of replicates to enhance statistical reliability of data obtained.
 8. The apparatus as set forth in claim 1 wherein said biologically-active-entity-responsive indicator means includes a nutrient medium for supporting the growth of a biologically-active entity to be assayed.
 9. The apparatus as set forth in claim 1 wherein said biologically-active entity responsive indicator means includes a diagnostic reagent giving rise to a visible manifestation upon presence of a biologically-active entity being assayed.
 10. The apparatus as set forth in claim 1 wherein size and geometry of said zones are such that some of said zones develop and others fail to develop manifestations of biological activity of entities present in the system to be analyzed.
 11. The method of separating a unitary sample into a series of aliquots and quantitatively assaying any system including liquids, solids, surfaces and gases to determine the number of biologically-active entities present in said system, said method comprising the steps of delineating a specific system to be analyzed, presenting to said system a sampling apparatus including means operative to extract and to retrieve From said system a series of aliquots in a single step, subjecting said aliquots to controlled environmental parameters including time and temperature conducive to promote a manifestation of biologically-active entities present in said aliquots, and examining said aliquots to determine the existence of indicia establishing the presence originally of biologically-active entities to ascertain the concentration of such biologically-active entities present in the system to be analyzed. 